Eomes pe

Manufactured by Thermo Fisher Scientific

Eomes-PE is a flow cytometry reagent that detects the expression of the Eomesodermin (Eomes) transcription factor. Eomes is a key regulator of T-cell and natural killer cell development and function. The Eomes-PE reagent can be used to identify and characterize Eomes-expressing cells in various cellular samples.

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6 protocols using eomes pe

Surface and Intracellular Staining of PBMCs

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Rested PBMCs from patients (n=25) and healthy controls were washed with PBS and surface stained for 30 minutes at 4°C. Monoclonal antibodies used for surface staining were LIVE/DEAD Aqua fixable dead cell stain (Life technologies), CD3-PerCP-Cy5.5 (clone HIT3a, BioLegend), and CD56-BV605 (clone NCAM16.2, BD Biosciences). Cells were then washed and incubated with BD Cytofix Fixation buffer for 20 minutes at 4°C. Once fixed, the cells were permeabilized in 1X Perm/Wash buffer (BD) for 20 minutes at room temperature and stained with T-bet-PE-Cy7 (BioLegend) and Eomes-PE (eBioscience). The cells were then washed and resuspended in 1X Perm/Wash buffer prior to analysis.

Bergerson R.J., Williams R., Wang H., Shanley R., Colbenson G., Kerber A., Cooley S., Curtsinger J.M., Felices M., Miller J.S, & Verneris M.R. (2016). Fewer circulating natural killer cells 28 days after double cord blood transplantation predicts inferior survival and IL-15 response. Blood Advances, 1(3), 208-218.

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Multiparameter Flow Cytometry of Immune Cells

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CD45.2-V500 and TNFα-APC-Cy7 were purchased from BD Biosciences (San Jose, California). KLRG1-FITC, Eomes-PE, CD107a-PE, CD107b-PE, CD27-PE, CD127-PE-Cy5, CD127-PerCP-Cy5.5, Tbet-PerCP-Cy5.5, IFNγ-PerCP-Cy5.5, Eomes-PErCP-efluor710, CD45.1-PECy7, KLRG1-PE-Cy7, Tbet-PE-Cy7, IRF4-AlexaFluor647, CD44-AlexaFluor700, CD62L-APC-eFluor780, CD44-eFluor450, KLRG1-eFluor450, IFNγ-eFluor450, CD90.2-APC-eFluor780, CD45.1-APC-eFluor780, IL-2-PerCP-Cy5.5 were purchased from eBioscience (San Deigo, California). CD8-PE-TexasRed, GranzymeB-PE, GranzymeB-APC, Live-Dead-Violet, Live-Dead-Aqua and goat-anti-rabbit IgG-AlexaFluor647 and -AlexaFluor488 were purchased from Life Technologies (Grand Island, New York). H2D^b-GP33 monomers were prepared at UMMS; LCMV-specific (H2D^b-NP396, H2D^b-GP276) and Influenza A PR8-OVA_I-specific (H2K^b-OVA257) monomers were obtained from the NIH Tetramer Core Facility (Atlanta, Georgia). Intracellular TCF1 staining was performed using rabbit-anti-mouse TCF1 (Cell Signaling Technology, Danvers, Massachusetts) followed by staining with goat-anti-rabbit secondary (Life Technologies). Samples were analyzed on an LSRII flow cytometer (Becton Dickinson), and data were analyzed using FlowJo (Tree Star).

Nayar R., Schutten E., Bautista B., Daniels K., Prince A.L., Enos M., Brehm M.A., Swain S.L., Welsh R.M, & Berg L.J. (2014). Graded levels of IRF4 regulate CD8+ T cell differentiation and expansion, but not attrition, in response to acute virus infection. Journal of immunology (Baltimore, Md. : 1950), 192(12), 5881-5893.

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T Cell Inhibitory Receptor Profiling in RA

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Peripheral blood mononuclear cells from HCs and paired PBMCs and SFMCs from RA patients were stained for the presence of T cell co-inhibitory receptors. Non-specific binding was blocked by 50 µg/ml mouse IgG (Jackson), and surface staining was performed using the following antibodies: CD3 V450 (clone: UCHT1, BD), CD4 PE-CF594 (clone: RPA-T4, BD), CD8 BV785 (clone: RPA-T8, BioLegend), CD25 Alexa 700 (clone: BC96, BioLegend), Tim-3 BV711 (clone: F38-2E2, BioLegend), CTLA-4 PerCPCy5.5 (clone: L3D10, BioLegend), Tigit PE-Cy7 (clone: MBSA43, eBioscience), PD-1 APC (clone: MIH4, BD), Live-dead near IR (Thermo Fisher Scientific). Cells were permeabilized by BD Facs lysing solution and BD Facs Perm Solution 2 (both BD bioscience). Intracellular staining was performed using Eomes PE (clone: WD1928 ebioscience). All antibodies were used in the concentration recommended by the manufacturer. Gating was done on lymphocytes, excluding doublets and dead cells. Gates were set using FMOs. CD3⁺ CD4⁺ and CD3⁺ CD8⁺ cells were investigated for their expression of T cell co-inhibitory receptors.

Greisen S.R., Yan Y., Hansen A.S., Venø M.T., Nyengaard J.R., Moestrup S.K., Hvid M., Freeman G.J., Kjems J, & Deleuran B. (2017). Extracellular Vesicles Transfer the Receptor Programmed Death-1 in Rheumatoid Arthritis. Frontiers in Immunology, 8, 851.

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Multiparameter Flow Cytometry Analysis

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The following antibodies were used (from BD Biosciences unless otherwise noted): CD8α-Pacific Blue or AlexaFluor (AF)700 (53-6.7, Biolegend); CD4 fluoroscein isothiocyanate (FITC) (GK1.5, eBiosciences), allophycocyanin (APC) (RM4-5), phycoerythrin (PE)-Cy5 or PE-Cy7 (RM4-5, Biolegend), or PE-TexasRed (RM4-5, Invitrogen), TCRβ APC-e780 (H57-597, eBiosciences), IL4Ra PE (mIL4R-M1), CD62L APC (MEL-14),, CD44 AF700 (IM7, Biolegend), CD122-biotin (TM-1) followed by streptavidin-PE Texas Red, Eomes-PE or AF647 (Dan11mag, eBiosciences), IFNγ-PerCPCy5.5 (XMG1.2, Biolegend), APC or PE, pSTAT6 PE (J1-773.58.11), and pAkt T308 PE (J1-223.371). For pS6 staining, cells were stained with a rabbit anti-phospoS6 monoclonal (2F9; Cell Signaling) and then stained with AF488 goat anti-rabbit IgG (Invitrogen). Live/Dead Aqua (Invitrogen) was used to exclude dead cells.

Carty S.A., Koretzky G.A, & Jordan M.S. (2014). Interleukin-4 Regulates Eomesodermin in CD8+ T Cell Development and Differentiation. PLoS ONE, 9(9), e106659.

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Multi-parameter Immune Cell Analysis

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Samples were thawed and incubated for 12-18 hours in RPMI media. For stimulation assays, CD3/CD28 beads (Life Technologies) were added 1:1 to the media during this incubation. Antibodies: L/D aqua (ThermoFisher), CD3-PreCP-Cy5.5, CD8-APC-C7, CD56-BV605 (BD), PD-1-FITC, Tim-3-BV711, TIGIT-APC (R&D Systems), CD45RA-AF700 (BD), CD27-PB, T-bet-PE-Cy7, Eomes-PE (eBioscience), IL-2-PE-Cy7, TNF-α-PE, and IFN-γ-PB. (BioLegend unless otherwise specified.) Cells were evaluated on BD LSR II flow cytometer and analyzed using FlowJo V10. Fluorescence minus one (FMOs) samples determined positive antibody expression.

Williams R.L., Cooley S., Bachanova V., Blazar B.R., Weisdorf D.J., Miller J.S, & Verneris M.R. (2017). Recipient T cell Exhaustion and Successful Adoptive Transfer of Haploidentical Natural Killer Cells. Biology of blood and marrow transplantation : journal of the American Society for Blood and Marrow Transplantation, 24(3), 618-622.

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Comprehensive T cell Immunophenotyping

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T cells from polarization assays were re-stimulated with 2 μL/mL cells of Cell Stimulation Cocktail (eBioscience), containing PMA/Ionomycin/Brefeldin-A/monensin, for 5 hours at 37°C. Cells were then stained with cell surface and intracellular antibodies using the Foxp3 Staining Buffer Set (eBioscience). Antibodies used for cell analyses were conjugated to FITC, PE, PerCp, or APC, and are as follows: CD4-PE, CD8-PErCp, CD25-FITC, CD44-PE, IFNγ-FITC, Annexin V-APC (from BD Biosciences),7-AAD (BD Pharmingen), granzyme B-FITC, T-bet-APC, Eomes-PE, RORγt-PE, IL-17A-APC (from eBioscience), and CD107-FITC (from BioLegend). For human CD8+ T cells, CD8-PE and IFNγ-PerCp-Cy5.5 (eBioscience) were used. Cells were analyzed on a BD FacsCalibur.

Glenn J.D., Smith M.D., Calabresi P.A, & Whartenby K.A. (2014). Mesenchymal stem cells differentially modulate effector CD8+ T cell subsets and exacerbate experimental autoimmune encephalomyelitis. Stem cells (Dayton, Ohio), 32(10), 2744-2755.

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